Novel regulatory elements of cold-inducible hutU gene from the Antarctic psychrotrophic bacterium Pseudomonas Syringae

ABSTRACT

A DNA sequence from the upstream region of cold-inducible hutU gene of the Antarctic Psychrotrophic Bacterium  Pseudomonas Syringae , comprising promoter elements and other regulatory sequences, with unique ‘CAAAA’ nucleotide sequence at −10 site of multiple transcription start sites and using said promoter to express genes of interest in the said bacterium at temperature as low as 4° C. and using the said bacterium with generation time ranging between two and half to three hours, as a system to produce low temperature labile proteins of pharmaceutical significance.

FIELD OF THE INVENTION

[0001] A DNA sequence from the upstream region of cold-inducible hutUgene of the Antarctic Psychrotrophic Bacterium Pseudomonas Syringae,comprising promoter elements and other regulatory sequences, with unique‘CAAAA’ nucleotide sequence at −10 site of multiple transcription startsites and using said promoter to express genes of interest in the saidbacterium at temperature as low as 4° C. and using the said bacteriumwith generation time ranging between two and half to three hours, as asystem to produce low temperature labile proteins of pharmaceuticalsignificance.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Antarctic bacteria provide a useful model system for studyingcold adaptation (15, 17, 31, 36). These organisms are generallyrepresented by the psychrotrophs and psychrophiles, which have theability to grow at 0° C. They can transcribe at this lower temperatureboth in vitro and in vivo (31). However, nothing much is known about thenature of promoter and regulatory elements from these bacteria or aboutthe mechanism of transcription at lower temperatures. Most of thetranscriptional studies thus far have been carried out with onlymesophilic bacterium, and the RNA polymerase from these bacteria,including Escherichia coli, cannot transcribe at 0° C. A recent studyfrom our laboratory has demonstrated that the RNA polymerase of theAntarctic psychrotrophic bacterium Pseudomonas syringae has cantranscribe at 0° C. The polymerase from the bacterium was not onlyactive at the low temperature but also could transcribe in vitropreferentially the cold-inducible gene of E. coli cspA from asupercoiled template (43). However, absolutely no information isavailable with regard to the characteristics of promoter sequence suchas the −10 and −35 elements from the bacterium for such low temperaturespecific transcription. Neither is any information available for the invivo recognition of promoter sequences by RNA polymerase from thecold-adapted P. syringae. Therefore, we initially attempted to identifythe genes from the Antarctic P. syringae that are upregulated at lowtemperature, with the help of TnJ-mediated random genomic fusions of aprompter-less reporter gene, lacZ (23). One of the fusions that producedat least 10- to 14-fcp,1d more p-galactosida.sc at a low temperature (4°C.) was identified by cloning and sequencing of ca. 450 bp of DNAsequence proximal to the Tn5 insertion site. The fusion was in the hutUgene, which encodes for an enzyme urocanase of the histidine utilizationpathway of bacteria (13, 23). A direct assay of urocanase activity fromthe P. syringae and a few more Antarctic Pseitdomonas species and theircomparison with the mesophilic P. putida suggested that the hutU gene isunregulated in the psychrotrophs but not in the mcso-phile. Therefore,it appeared to us that the hutUgene might be a useful model forinvestigating the mechanism of gene regulation at low temperatures inthe Antarctic bacterium. Accordingly, we cloned and sequenced the DNAencompassing the hut if gene, and its upstream and downstream regionsand identified different open reading frames (ORFs) in the region. Wealso examined transcripts from bacterial cells grown at low (4° C.) andhigh (22° C.) temperatures by Northern and primer extension analyses,and we identified the transcription start sites and other putativeregulatory elements of the hutU gene. Additionally, we compare here thededuced amino acid sequences of the urocanase from the psychrotrophic P.syringae and other bacteria, including the mesophilic P. putida, inorder to examine the possible amino acid substitutions due to alow-temperature adaptation.

OBJECTS OF THE PRESENT INVENTION

[0003] The main object of the present invention is to determine the roleof promoter and other regulatory elements of antarctic psychotrophicbcterium pseudomonas syringae in expression of proteins under extremelylow temperature.

[0004] Another object of the present invention is to express theproteins in the said system at fast rate.

[0005] Yet another object of the present invention is to determine thetranscription initiation site.

[0006] Still another object of the present invention is to determine thetranslation start site.

[0007] Still another object of the present invention is to determinesequence homology of regulatory elements.

[0008] Still another object of the present invention is to develop amethod of producing said sequence from said bacterium.

[0009] Still another object of the present invention is to develop amethod of using the said sequence to produce heat labile proteins.

[0010] Still another object of the present invention is to develop amethod of using the said sequence to produce heat labile proteins ofpharmaceutical importance.

SUMMARY OF THE PRESENT INVENTION

[0011] A DNA sequence from nucleotide 2961 to 3600 of the upstreamregion of cold-inducible hutU region of the Antarctic PsychrotrophicBacterium Pseudomonas Syringae of accession No.AF326719, comprisingpromoter elements and other regulatory sequences, with unique ‘CAAAA’nucleotide sequence at −10 site of multiple transcription start sitesand using said promoter to express genes of interest in the saidbacterium at temperature as low as 4° C. and using the said bacteriumwith generation time ranging between two and half to three hours, as asystem to produce low temperature labile proteins of pharmaceuticalsignificance.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0012] A DNA sequence from nucleotide 2961 to 3600 of the upstreamregion of cold-inducible hutU region of the Antarctic PsychrotrophicBacterium Pseudomonas Syringae of accession No.AF326719, comprisingpromoter elements and other regulatory sequences, with unique ‘CAAAA’nucleotide sequence at −10 site of multiple transcription start sitesand using said promoter to express genes of interest in the saidbacterium at temperature as low as 4° C. and using the said bacteriumwith generation time ranging between two and half to three hours, as asystem to produce low temperature labile proteins of pharmaceuticalsignificance.

[0013] In an embodiment of the present invention promoter has twotranscription initiation sites with low temperature (4° C.), and commonlow and high temperature (4° C. and 22° C.) specificity.

[0014] In yet another embodiment of the present invention the amount oftranscripts produced at 4° C. from the hutU gene and hut operon is about20-fold higher than the amount present at 22° C. during the steady-stategrowth of the said bacterium.

[0015] In still another embodiment of the present invention the saidgene is inducible upon a downshift of temperature from 22 to 4° C.

[0016] In yet another embodiment of the present invention HutU is theOpen Reading Frame (ORF) of hutU gene.

[0017] In still another embodiment of the present invention amount ofmRNAs from the hutU operon increased only about two to three folds ontemperature downshift of the said bacterium from 22 to 4° C.

[0018] In yet another embodiment of the present invention amount oftranscripts produced at 4 and 22° C. or from “cold-shocked” cells aftera shift of the culture from 22 to 4° at different time points of 0, 0.5,1, 2, and 3 hours after that shift.

[0019] In still another embodiment of the present invention the amountof mRNAs is at maximum by 2 hours after the shift and decreasedsubsequently.

[0020] In yet another embodiment of the present invention 4° C. specifictranscription start site starts with a G, which is 219 nucleotidesupstream of the translation initiation codon GTG of the HutU ORF.

[0021] In still another embodiment of the present invention the commontranscription start site for both low and high temperature is located 39nucleotides from the low temperature transcription start site.

[0022] In yet another embodiment of the present invention the −35sequence in the promoter region of the 4° C. specific transcript isTGTTAC.

[0023] In still another embodiment of the present invention the −35sequence in the promoter region of the 4 and 22° C. common transcript isCCTGCG.

[0024] In yet another embodiment of the present invention the 4° C.specific transcript has second CAAAA sequence at the −15 position.

[0025] In still another embodiment of the present invention the secondCAAAA nucleotide sequence at low temperature transcript is important forincreased expression of the gene at lower temperature.

[0026] In yet another embodiment of the present invention sequence atthe upstream of the GTG translational start codon of the hutU genecontain HutC repressor binding motif CTTGTATGTACAAG.

[0027] In still another embodiment of the present invention cataboliteactivator protein (CAP) binding sequence, AAGTGTGCGTCGACCCTCTTGT, islocated 35 nucleotides upstream of the GTG translation initiation codonof the hutU-ORF.

[0028] In yet another embodiment of the present invention saidCatabolite Repressor acts as transcriptional roadblock for RNApolymerase.

[0029] In still another embodiment of the present invention a conserved“cold-box”-like sequence, TTGATGAACAACC, is located 123 nucleotidesdownstream of the translation initiation codon of HutU-ORF.

[0030] In yet another embodiment of the present invention nitrogenregulatory σ^(N) promoter element, GGCCGCTTACTTGC, is located 81nucleotides upstream of the translation start site of the HutU-ORF.

[0031] In still another embodiment of the present invention said HutUgene can not be expressed in E. coli at temperature of about 15° C. andbelow.

[0032] In yet another embodiment of the present invention theShine-Dalgarno (SD) sequence, GAGGA, is located 12 nucleotides upstreamof the translation initiation codon GTG of the hutU ORF.

[0033] In still another embodiment of the present invention thecold-shock protein binding sequence, ATTGG, is located 186 nucleotidesdownstream of the translational initiation codon GTG of hutU ORF.

[0034] In yet another embodiment of the present invention regulatorysequence GCGAGCTCTTGAATGCGGCCACCAAGAGCTCGC, having hairpin loopstructure is located 70 nucleotides upstream of low temperaturetranscription start site. In still another embodiment of the presentinvention change of free energy (ΔG) for the said loop is about −16.6kcal.

[0035] In yet another embodiment of the present invention said loopfunctions as a transcription stop signal for the upstream hutC gene.

[0036] In still another embodiment of the present invention said loopfunctions as a regulatory element for transcription of the hutU gene.

[0037] Another embodiment of the present invention, a method of cloningand expressing cold-inducible hutU gene from the Antarctic PsychrotropicBacterium Pseudomonas Syringae, said method comprising steps of:

[0038] a. cloning 2.4 kbp Pst DNA fragment containing hutU gene, andthree overlapping fragments containing upstream 3.5 kbp Eco-RI-Kpn Ifragment, downstream 2.54 Kbp SalI and 1.2 Kbp Pst I fragments intopUC19,

[0039] b. sequencing the clone, and

[0040] c. expressing the sequence clone.

[0041] In yet another embodiment of the present invention expressinggene encoding low-temperature labile proteins of interest using DNAsequence of claim 1, said method comprising introducing said sequence atthe upstream region of the gene, and expression the said gene, obtainingprotein of interest.

[0042] A promoter-fusion study with a Tn5-based promoter probe vectorhad earlier found that the hutU gene which encodes the enzyme urocanasefor histidine utilization pathway is unregulated at a lower temperature(4° C.) in the Antarctic psychrotrophic bacterium Pseudomonas syringae.To examine the characteristics of the uroranase gene and its promoterelements from the psychrotrophs, the complete hutU and its upstreamregion from P. syringae were cloned, sequenced, and analyzed in thepresent study. Northern blot and primer extension analyses suggestedthat the hiitU gene is inducible upon a downshift of temperature (22 to4° C.) and that there is more than one transcription initiation site.One of the initiation sites was specific to the cells grown at 4° C.,which was different from the common initiation sites observed at both 4and 22° C. Although no typical promoter consensus sequences wereobserved in the flanking region of the transcription initiation sites,there was a characteristic CAAAA sequence at the −10 position of thepromoters. Additionally, the location of the transcription andtranslation initiation sites suggested that the hutU mRNA contains along 5′-untranslated region, a characteristic feature of manycold-inducible genes of mesophilic bacteria. A comparison of deducedamino acid sequences of urocanase from various bacteria, including themesophilic and psychrotrophic Pseudomonas sp., suggests that there is ahigh degree of similarity between the enzymes. The enzyme sequencecontains a signature motif (GXGX₂GX₁₀G) of the Rossmann fold fordinucleotide (NAD*) binding and two conserved cysteine residues in andaround the active site. The psychrotrophic enzyme, however, has anextended N-terminal end.

[0043] Materials and Methods

[0044] Bacterial strains and growth conditions. The Antarcticpsychrotrophic bacterium P. syringae Lz4W, which grew optimally at 22°C. was isolated, identified, and maintained as reported earlier (42).Routinely, the culture was grown on antarctic bacterial medium, whichcontains 0.5% peptone (wt/vol) and 0.2% (wt/vol) yeast extract, at roomtemperature (22° C.) or at a cold temperature (4° C.) when needed. The£. coli cells were grown at 37° C. in Luria-Bcrtani (LB) medium andmaintained on LB agar plates.

[0045] DNA manipulation techniques and cloning of hulU gene. Thebacterial genomic DNA was prepared by as described previously (32).Isolation of plasmid DNA, restriction endonuclcase digestion, ligationtransformation, and agarose gel electrophoretic separation of DNA werecarried out as described by Sam-brook ct ul. (39).

[0046] For cloning of the hulU gene from P. syringae, a 522-bp DNAfragment from the plasmid pF43, which contained the hulU proximal regionof the promoter fusion clone F43 (23) was used as a probe. Initially, a2.4-khp ftrl DNA fragment containing the hulU gene was cloned in pUC19(phlSO). Subsequently, three more overlapping fragments containing theupstream 3.5-kbp EcoRl-Kpnl fragment (phUp) and the downstream 2.54-kbpSail and 1.2-kbpft/l fragments (ph20 and ph39, respectively) werecloned. A total of 6.578 kbp were thus cloned spanning the region (FIG.1A).

[0047] SNA sequence analysis. Nuclcotidc sequence determination werecarried out on an automated DNA sequencer (ABI model 377), by usingdouble-stranded plasmid DNAs as a template and ABI PRISM Dye terminatorcycle sequencing method (Pcrkin-Elmcr). The BLAST programs (3.4) wereemployed for DNA sequence homology search in the NCBI GcnBank sequencedatabase (hup://www.ncbi.nlm.gov/). PCGENE programs were also used forvarious DNA and amino acid sequence analyses. Secondary structureprediction analysis of protein was carried out by using thePrcdict-Protcin PHD mail server at EMBL. Heidelberg(predict-HclpfffEMBL-Heidclbcrg.DE), which uses combined evolutionaryinformation and neural networks for structural predictions (34, 35). Thenucleotide sequence reported here has been submitted to GcnBank underaccession number AF326719.

[0048] Isolation nf RNA and Northern analysis. RNA was isolated by hotphenol method (1) from the bacterial cells (optical density at 600 nm of−0.5) grown at 22 and 4° C. For the isolation of RNA from cold-shockedcells, P. syringae was initially grown at 22° C. and then shifted to 4°C. and incubated for various time periods before the isolation. Northernhybridization was carried out as described earlier (32) but with thehulU- and hulP-spcciftc probes. Dcnsitometer scanning (MolecularDynamics) of the autoradiograms was carried out for estimating the foldinduction of RNA.

[0049] Primer extension analysis. Primer extension analyses were carriedout by two methods on the total RNA isolated from 4 and 22° C. growncells. In the first method (39), a ^(1::)P-cnd-labclcd primer(5′-GGGTAGTTGAAGTCACGGGTA AC-3′) corresponding to the complementarysequence of the putative N-terminal end of the HutU-ORF (−7 to +16nuclcotidcs with respect to the GTG initiation codon) of P. syringae wasextended in the presence of Moloncy murinc leukemia virus reversetranscriptase (Pharmacia). In the second method, the unlabeled primerwas extended in the presence of |a-³²P|ATP and Moloncy murinc leukemiavirus reverse transcriptase as described earlier (8). Both methodsproduced similar results, except that the primer-extended products weresharper with less background on the autoradiograms obtained by thesecond method. DNA sequencing reactions were also carried out with thesame primer on the double-stranded DNA template (phlSO) with a Scquenasc2.0 Kit (U.S. Biochcmicals) and run in parallel with the primer extendedproducts on an 8 M urca-6% polyacrylamidc gel for mapping thetranscriptional start points as described previously (39).

[0050] Enzyme assays. Urocanasc activity was assayedspcctrophotomctrically by the method of George and Phillips (14).3-Galactosidasc was assayed by the method of Miller (28). Proteins wereestimated by the method of Bradford (10).

BRIEF DESCRIPTION OF THE ACCMPANYING DRAWINGS

[0051]FIG. 1 Shows Physical map and the organization of the hutU regionof P. syringae. (A) A 6.578-kbp DNA from the hutU region of P. syringaewas sequenced and analyzed by cloning the region on four overlapping DNArestriction fragments (phUp, phlSO, ph20, and ph39) shown below thephysical map. Only the major restriction enzyme recognition sites (E,EcoRI. K, Kpnl, P, ft/I: S, Sail, Sm, Smal) are indicated on thephysical map. The genes (sdeB, hutC, hutU, and hutP) and theirdirections of transcription are shown at the top. The putative ORFs.such as SdeB, HutC-ORFI and -ORFII, HutU and HutP are shown as shadedboxes below'the physical map. The incomplete ORF of HutP is indicated asa box with a slope at the C-terminal end. A hairpin structure betweenHutC and HutU indicates the location of a dyad structure in the DNAsequence. (B) The organization of the genes in the hutU region of a fewbacteria identified either by earlier genetic studies or by recentgenome sequence analyses are shown for comparison. The directions oftranscription are indicated above the genes by arrows. The genesencoding various enzymes involved in histidine utilization (hut) areabbreviated as hutH (for histidase), hutU (for urocanase), hull (forimidazolone propionate hydrolase), hutF (for FIGLUase), huiG (forformylglutamate amidohydrolase), hutT (for inducible histidine/urocanatetransporter), hutP (putative transporter with similarity topurine-cytosine permease), hutC orfl (putative re-pressor of hutoperon), and hutC orfll (3′ downstream ORF of hutC). The unknown ORFs ofthe hutU region in some bacteria are indicated directly by their ORFidentification numbers (e.g., VC1201, VC1207, 3A, SC25.10, SC25.15C, andDRA0146).

[0052]FIG. 2 Shows Multiple sequence alignment of the deduced amino acidsequence of urocanase and the predicted secondary structure of theenzyme. The sequences of the hiitU genes (the numbers in parentheses areGenBank accession numbers) include: P. pittida (M33923), P. aeruginosa(AE004922), A. rhizo %enes (AB039932), T. rcpens (P53385), V. cholerae(AE004200), Y. pcstis (AL031866), B. subtilis (P25503), 5. coelicolor A3(AL354048), and D. radiodurans (AE001862). The CLUSTAL X (1.8) Programwas used for the alignment. The amino acids have been shown in singleletter code. The characters (“#” and “:”) indicate the positions ofperfectly conserved amino acids and substitution by similar amino acids,respectively. A period indicates the position is conserved in mostorganisms. The highly conserved active site of the enzyme (FQGLPARICW)is shown within a box. Apart from the conserved cysteine within theactive-site box, a second conserved cysteine (C-363 of P. syringaecorresponding to C-355 of the mesophiiic P, putida) that is importantfor enzyme activity (25) has also been marked. The predicted secondarystructure shown above the aligned sequences is based on PHDs (profilefed neural network system) developed by Rost et al. (34, 35) and foundat predict-Help (o>EMBL-Heideiberg.DE. The helices have been shown ascylinders, the (3-sheets are indicated as block arrows, and the otherstructures, including the loops and coils, are appear as thick straightlines. The Rossman fold for dinucleotide binding sequence GXGX₂G-X₁₀-G(where X is a nonspecific amino acid) has been marked below the alignedsequences. A 3-slieet of “SLNIE” following the helix of Rossman fold isalso highly conserved, which contains an acidic residue (E or D) at the3′ end of the sheet that is generally specific for NAD*. Also note thata predicted short N-terminal helix (aa residues 10 or 13) is presentonly in P. syringae among the compared organisms.

[0053]FIG. 3 Relationship among bacterial urocanases. Similaritiesbetween the enzymes have been shown as an unrooted neighbor-joiningtree, giving all branch lengths (indicated by numerical values). Thetree has been drawn by a neighbor-joining plot of the CLUSTAL X (version1.8) program. The urocanase sequences of gram-negative bacteria (e.g. P.syringae, P. putida. P. aeruginosa, V cholerae, and A. rhizogenes) andgram-positive bacteria (e.g., B. subtilis, S. coelicolor, and D.radio-ilnrans) fall into two distinct clusters and may have divergedearly in evolution. The only sequence of possible plant origin (33),from T. repens, is closer to the sequence of gram-negative bacteria.

[0054]FIG. 4 Northern analysis of transcripts from the HutU region in P.syringae. The RNAs were isolated either from the cells exponentiallygrowing at 4 and 22° C. or from “cold-shocked” cells after a shift ofthe culture from 22 to 4° C. at different time points (0, 0.5, 1, 2. and3 h) after that shift. The transcripts from the hutU region are shown ata steady-state level (a) and during the cold-shock induction (b). (c)The RNA loading control (10 u.g) used in the study is shown in acthidium bromide-stained gel. The positions of the 23S and 16S rRNAs andof the 4S RNA are marked by arrows. The positive signals due to thehybridization of a ′^(:)P-labeled 2.4-kbp DNA fragment probe spanningall offiutU and part ofhutP (derived from phi80) are marked byarrowheads.

[0055]FIG. 5 Primer extension analysis of tuttU transcripts. The RNAsprepared from cells of P. syringae grown at 22 and 4° C. were used forprimer extension reactions in the presence of Moloney murine leukemiavirus reverse transcriptase and [a-³²P]ATP, as described in Materialsand Methods. The products were analyzed on a sequencing gel (8 M urea-6%polyacrylamide gel electrophoresis) in parallel with the sequencingreaction products (i.e. A, C, G, and T) carried out with the sameprimer. The major primer-extended products are marked by arrowheads.Note that the two longest primer-extended products were considered foranalyzing the promoters in the present study (see FIG. 6). The 4° C.specific longest product was found repeatedly absent in the 22° C. RNAspecific reactions. The shortest product (the lowermost arrow) wascompatible with a putative (T⁵⁴-specific promoter located in theupstream region of the HutU gene.

[0056]FIG. 6. (A) DNA sequence from the upstream region of hutU of P.syringae containing the promoter elements and other putative regulatorysequences. The dashed arrow shown below the sequence at beginning of theAurtAspecific ORF was used for mapping the transcription start sites.The temperature (4° C. and 4 or 22° C.)-specific transcription startsites have been indicated by shaded arrows. The characteristic CAAAAsequence in the promoters is shown within the boxes. The putative(7³⁴-specific promoter sequence [GG(N|₀)-GG] and the correspondingtranscription start site (vertical open arrow) is marked on thesequence. The putative repressor HutC binding sequence (CTTGTATGTACAAG)and CAP binding sequence (AAGTGTGCGTCGACCCTCTTGT) have been marked byshaded boxes. The translation initiation codon (GTG), the Shine-Dalgarno(SD) sequence, a putative cold box-like sequence, and a cold-shockprotein binding sequence (ATTGG) downstream of the translationalinitiation site of hutU are marked. The sequence of the 3′ end of theorfl of the hutC are underlined and in lowercase. The nucleotide numbersrefer to the number in the DNA sequence of the region from P. syringae(accession no. AF326719). (B) Putative regulatory sequence with thepotential hairpin structure in the promoter region of hutU. The locationof the hairpin is marked by two opposing arrows below the sequence inpanel A.

RESULTS

[0057] Cloning and sequence analysis of the hutU region from P.syringae. A 522-bp Tn5-proximal sequence of the hutU promoter fusionclone (F43) of P. syringae (23) was used as a probe to clone the wholeof the hutU gene and its upstream and downstream sequences, as describedin Materials and Methods. The reported region (6.578 kbp) was cloned onthree overlapping DNA fragments (FIG. 1A). The DNA sequence of theregion were determined (accession no. AF326719) and analyzed by a BLASTsearch (3, 4). Four complete and one incomplete ORFs were identifiedwhich had homology with the hypothetical sdeB gene homologue of Yersiniapestis (accession no. AL031866), hutC (ORFI), hutC (ORFII), and hutU AQgenes of the hut operon of P. putida (2, 13, 19) and the gene yxIAhomologue for purine cytosine permease of Bacillus subtilis (accessionno. E70081), respectively (FIG. 1). At the level of amino acids, theSdeB homologue (456 amino acids [aa]) was 68% similar to the Y pestissequence, and the HutC ORFI (249 aa), HutC ORFII (192 aa), and HutU (565aa) were 91.63, and 93% similar, respectively, to the P. putidasequences. The ORF of the last gene (yxIA homologue) was incomplete (433aa versus the complete 457 aa of YxIA), as shown in FIG. 1A. Thedirection of transcription of the sdeB was opposite to the direction ofthe other genes. When this gene organization was compared with theorganization of hut operon of the mesophilic P. putida (19) it wasobserved that the relative positions oihutC (orfl), hutC (oiflf) andhutU in these two bacteria were identical. The difference was noticed inthe hutU downstream region of P. syringae, which was occupied by ahomologue to the gene for purine-cytosine permease (henceforth referredto as hutP) of B. subtilis and yeast, instead of the hutH (encodinghistidasej found in F. putida (FIG. 1B). An electronically submitteddata (accession no. AF032970) reveals that a homologue of hutT (encodingan inducible urocanate transporter) is present in the downstream ofhutHin P. putida, which was not earlier identified by the genetic study(19, 20). The genome sequence data of P. aeniginosa(http://www.pseudomonas.com) reveals that the downstream of thshutU genein the bacterium (FIG. 1B) contains the homologues for both hutP (genefor purine-cytosine permease) and hutT (gene for urocanate transporter).The importance and functional significance of the occurrence of apurine-cytosine permease homologue in the hut operon of P. syringae isnot clear at present. However, from a comparative analysis of the genessurrounding the hutU region of different bacteria (FIG. 1B), it isapparent that the organization of the genes in the hutU region is quitedivergent among various bacteria.

[0058] It is also interesting that the genetic data of P. putida hadearlier established the occurrence of the hutF gene (encoding the enzymeformiminoglutamate iminohydrolase [FIGLUase]) at the upstream of thelititC (19, 20). The hutF gene was shown transcribed divergently fromthe hutC, similar to the direction of transcription of the sdeBhomologues observed in the genome sequence of many gram-negativebacteria, such as P. syringae, P. aeniginosa. Agrobacterium rhizogenes.and Y pestis (FIG. 1B). Since the DNA sequence for the hutF gene is notknown from P. putida or any other bacterium, it remains to be determinedwhether the sdeB homologue, which also has similarity with the atrazinechlorohydrolases (AtzA, 23% identity; accession no. U55933.1) and themelamine deaminase (TriA, 23% identity; accession no. AF312304.1) ofPseudomonas species is the hutF gene of these gram-negative bacteria. Inthis context, it is interesting that the DNA sequence 108 bp upstream ofliutC region of P. putida (accession no. M33922) contains a beginning ofa divergently transcribed ORF (N-terminal 19 aa), which is 93% similarto the first 19 aa of the sedB homologue tjf P. syringae. It is alsoimportant to note that the hutF is generally thought to be unique forPseudomonas species. The two genes, hutF and hutG, encoding FIGLUase andformylglutamate hydrolase, respectively, are involved in the catalyticconversion of formiminoglutamate (FIGLU) to glutamate and formate by atwo-step process in pseudo-monads. This is in contrast to otherbacteria, such as Klebsiella and B. subtilis, where a single enzymeencoded by hutG catalyzes the conversion of FIGLU to glutamate andformamide at the final step of the histidine degradation pathway (19,26) amino acids, the SdeB homologue (456 amino acids [aa]) was 68%similar to the Y. pestis sequence, and the HutC ORFI (249 aa), HutCORFII (192 aa), and HutU (565 aa) were 91, 63, and 93% similar,respectively, to the P. putida sequences. The ORF of the last gene (yxlAhomologue) was incomplete (433 aa versus the complete 457 aa of YxlA),as shown in FIG. 1A. The direction of transcription of the sdeB wasopposite to the direction of the other genes. When this geneorganization was compared with the organization of hut operon of themeso-philic P. putida (19) it was observed that the relative positionsof hutC (orff), hutC (<<///)• ^(an£)1 Aitft/in these two bacteria wereidentical. The difference was noticed in the ItutU downstream region ofP. syringae, which was occupied by a homologue to the gene forpurine-cytosine permease (henceforth referred to as hutP) of B. subtilisand yeast, instead of the HutH (encoding histidase) found in f″. putida(FIG. 1B). An eiectronically submitted data (accession no. AF032970)reveals that a homologue of hutT (encoding an inducible urocanatetransporter) is present in the downstream of liutH in P. putida, whichwas not earlier identified by the genetic study (19, 20). The genomesequence data of P. aeruginosa (http://www.pseudomonas.com) reveals thatthe downstream of the/wri/gene in the bacterium (FIG. 1B) contains thehomologues for both hutP (gene for purine-cytosine permease) and hutT(gene for urocanate transporter). The importance and functionalsignificance of the occurrence of a purine-cytosine permease homologuein the hut operon of P. syringae is not clear at present. However, froma comparative analysis of the genes surrounding the hutU region ofdifferent bacteria (FIG. 1B), it is apparent that the organization ofthe genes in the hutU region is quite divergent among various bacteria.

[0059] It is also interesting that the genetic data of P. putida hadearlier established the occurrence of the hutF gene (encoding the enzymefomiminoglutamate iminohydrolase [FIGLUase]) at the upstream of the hutC(19, 20). The hutF gene was shown transcribed divergently from the hutC,similar to the direction of transcription of the sdeB homologuesobserved in the genome sequence of many gram-negative bacteria, such asP. syringae, P. aeruginosa. Agmbacterium rhizogenes. and Y pestis (FIG.1B). Since the DNA sequence for the hutF gene is not known from P.putida or any other bacterium, it remains to be determined whether thesdeB homologue, which also has similarity with the atrazinechlorohydrolases (AtzA, 23% identity; accession no. U55933.1) and themelamine deaminase (TriA, 23% identity; accession no. AF312304.1) ofPseudomonas species is the hutF gene of these gram-negative bacteria. Inthis context, it is interesting that the DNA sequence 108 bp upstream ofhutC region of P. putida (accession no. M33922) contains a beginning ofa divergently transcribed ORF (N-terminal 19 aa), which is 93% similarto the first 19 aa of the sedB homologue of P. syringae. It is alsoimportant to note that the hutF is generally thought to be unique forPseudomonas species. The two genes, hutF and hutG, encoding FIGLUase andformylglutamate hydrolase, respectively, are involved in the catalyticconversion of formiminoglutamate (FIGLU) to glutamate and formate by atwo-step process in pseudo-monads. This is in contrast to otherbacteria, such as Kkbsiella and B. subtilis, where a single enzymeencoded by hutG catalyzes the conversion of FIGLU to glutamate andformamidc at the final step of the histidine degradation pathway (19,26).

[0060] Predictive analysis of the amino acid sequence of urocanase fromP. syringae. In recent times comparative analyses of proteins frompsychrophilic, mesopnilic, and thermophilic bacteria have shed new lighton the structural adaptability of the enzymes for catalysis at lowtemperatures and has provided some clues as to their thermal stability(15). Therefore, it was interesting to compare the primary sequence ofthe urocanase from the psychrotrophic and mescphilic bacteria.Accordingly, the deduced amino acid sequences of the enzyme from thepsychrotrophic P. syringae and mesophilic P. putida were compared (FIG.2). It was observed that the P. syringae enzyme R contains an overallidentity of ca. 90% (similarity of 93%) to the P. putida enzyme but hadan additional 8-aa extension at the N-terminal end (making it 565 aalong). A BLAST search analysis picked up the urocanase homologues fromvarious other bacteria for which the fuitU gene sequences are known. Analignment of the deduced amino acid sequences of the enzyme from thesebacteria is shown in FIG. 2. Although the enzyme seems to be quiteconserved, it fell into two distinct groups of gram-positive (B.subtilis, Streptomyces coelicolor, and Dt′inococcus radiodurans) andgram-negative (P. syringae. P. putida, P. aentginosa. Vibrio cliolerae.and A. rliizogenes) \Q: strains (FIG. 3). The urocanase from the plant(Trifblium re-re pens) was found clustered with the enzymes fromgram-negative bacteria.

[0061] From the compositional analyses of the amino acids of urocanaseit was apparent that the contents of isoleucine plus leucine (13.1%),the aromatic amino acids (7.2%), and proline (4.4%), which are generallyknown to change in the psychrophilic enzymes (15), were similar in P.syringae and mesophilic P. putida (13.28, 7.5, and 4.4%, respectively).A slight increase in the content of serine and threonine (10.4%) was,however, noticed in P. syringae when it was compared with the enzymesfrom P. putida and other organisms (7.98 to 9.5%). The argi-nine content(5.3%), which is known to stabilize the helices, was also marginallylower in P. syringae than in the P. putida enzyme (5.9%). The P.syringae enzyme also contained eight cysteines compared to the sevencysteines of P. putida and P. aeruginosa. Six-of these cysteines arcconserved among them and are located in equivalent positions, includingthe two important cysteines C-410 and C-354 (C-419 and C-363 in P.syringae), which were shown to be involved in catalysis and substratebinding, respectively, in the enzyme from P. putida (25). Interestingly,the radiotolerant bacterium D. radiodurans contains only these twoconserved cysteines in the enzyme.

[0062] Predictive structural analyses of the urocanase sequence from P.syringae exhibited some interesting features (FIG. 2). The enzymecontained a conserved P-a-p structural motif of the dinucleotide bindingRossman fold (GXGX₂G-X-G/A) for NAD* binding at the 182- to 198-aaregion. A T blast analysis suggested that this NAD binding region hasstructural homology with the similar region of the glutamatedehydroge-nase, including an acidic amino acid residue (E-205 in thecase of P. syringae urocanase) at the end of the p-strand (²⁰¹SLNIE²⁰⁵)of the β-α-βmotif (7). The acidic amino acid residue commonly forms thehydrogen bonds to the adcnine ribose hydroxyls and is generally thoughtto be an indicator of NAD+ specificity as opposed to the NAD+specificity (6, 7). An implication of the identification of the Rossmanfold in urocanase would be that, although the loop is similar to that ofother NAD+ binding proteins, it has to be juxtaposed within a tightlyheld tertiary or quaternary structural fold of the protein. This isbecause the exogenously added NAD could not be incorporated into theenzyme in vitro, nor could the coenzyme be dissociated from the proteinwithout irreversible denaturation of the protein (33). The presentpredictive analysis (FIG. 2) also suggests that the P. syringae enzymecontains at the N-terminal end a short a-helix that is absent in otherbacteria. Temperature-dependent expression of hutU in P. syringae.Northern analyses were carried out to examine the expression of the hutUgene in P. syringae at low (4° C.) and high (22° C.) temperatures. Itwas observed that, during the steady-state growth of the bacterium, theamount of transcripts produced at 4° C. from the hutU gene and hutoperon was ca. 20-fold higher than the amount present at 22° C. Threetranscripts (ca. 7.1, 2.1,and 1.0 kb) hybridized to the probe of 2,4-kbpPstl fragment containing both hutU and hutP genes (FIG. 4a). ThehutP-F<specific probe, however, hybridized only to the 7.1-kb transcript(data not shown). Thus, it would appear that the 7.1-kb transcript mightrepresent the polycistronic mRNA, whereas the 2.1- and 1.0-kbtranscripts represent the processed and/or degraded product from theoperon. The 2.1-kb transcript can potentially encode the full-lengthhutU gene and therefore might be physiologically important.

[0063] Interestingly, upon a temperature downshift of the bacterium from22 to 4° C., a “cold-shock” response was noticed because the amount ofmRNAs from the hut operon increased only about a maximum of two- tothreefold after the shift (FIG. 4b). The amount of the mRNAs was at amaximum by 2 after the shift and decreased subsequently. Thus, itappears that the hut operon might have cold-responsive regulatoryelements that are known to occur in cold-inducible genes of mesophilicbacteria (12, 29).

[0064] Transcription start site and promoter region of the hutU gene ofP. syringae. Primer extension analyses were carried out to locate thetranscription start sites of the hutU gene, with the RNAs isolated fromcells of P. syringae grown at low (4° C.) and high (22° C.) temperatures(FIG. 5). Among several primer-extended products, the longest product(topmost arrow in FIG. 5) was repeatedly observed only with the RNAsisolated from the cells grown at 4° C. Most of the other extendedproducts were common in cells grown at both both low and hightemperatures. Based on the longest primer-extended products, thetranscription start sites for the low temperature (4° C.) and the commonsite for both low and high temperatures (4 and 22° C.) were located inthe 5′ region of the hutU gene (FIG. 6A). Rest F6 of the smallerextended products of the primer extension reactions (FIG. 5) couldeither represent the true transcription start sites or the processed ordegraded ends of ttie mRNAs.

[0065] The low-temperature (4° C.) specific transcript starts with a G,which is 219 nucleotides upstream of the putative translation initiationcodon GTG of the HutU ORF. The common transcription start site for bothlow and high temperatures is located 39 nucleotides downstream from theformer start site (FIG. 6A). Thus, the transcripts from the hut operonseems to have a long 5′ untranslated region (5′-UTR) sequence that is acharacteristic of the cold-shock genes in mesophilic bacteria, including£. colt (12). Upon further examination of the DMA sequences around thetranscription start sites, it was observed that the −10 region has acharacteristic CAAAA sequence at both temperatures. The −35 sequences inthe promoter region of the 4° C. specific and the 4 and 22° C. commontranscripts were TGTTAC and CCTGCG, respectively. Interestingly, thepromoter region of the 4° C. specific transcript had a second CAAAAsequence at the −15 position; the significance of this, if any, is notyet clear.

[0066] The sequence at the upstream of the GTG translational start codonof the hutU gene of P. syringae contained a putative HutC represserbinding motif (CTTGTATGTACAAG), which is slightly different from thesequence observed in P. putida and Klebsiella aerogenes (2, 40).Interestingly, in the latter organisms this sequence overlaps with the−35 element of the promoter region, in contrast to the case in P.syringae, which contains the sequence within the 5′-UTR of the gene(FIG. 6A). It was also observed that a putative nitrogen assimi-latorycofactor-binding sequence [ATA-(N_(A))-TAT] is located overlapped withthe HutC-binding motif (41). A putative catabolite activator protein(CAP) binding sequence was also noticed in the downstream oftranscription initiation site. The sequence, AAGTGTGC(N_(ft))CCTCTTGT,is located at the 35 nucleotides upstream of the GTG translationinititation codon of the hutU-ORF. In K. aerogenes a similar sequencewas observed centered around nucleotide −81.5 of the promoter region ofImtUH gene cluster (30). Since the CAP binding sequence occursdownstream of the transcription initiation site of P. syringae, thecatabolite represser in this case could act in theory as atranscriptional roadblock for RNA polymerase. It was also noticed that aputative conserved “cold-box”-Iike sequence (TTGATGAACAACC), whichoccurs at the 5′ end of the 5′-UTR of the cold-inducible genes of E.coll, is located 123 nucleotides downstream of the translationinitiation codon of HutU in P. syringae (FIG. 6A). Since the location ofthe cold box is at variance with E. coli, any functional significance ofthe element in P. syringae remains to be determined. A putative nitrogenregulatory o^(−N) promoter element [GG-(N₁₀)-GC] was also noticed at the81 nucleotides upstream of the-translation start site of the HutU-ORF ofP. syringae. This is interesting for the observation that the shortestprimer-extended product (lowest arrow in FIG. 5) is located very closeto the expected position of the transcription start site (at the −13position rather than at the expected −12 position of the initiationsite) of the above o^(−N) promoter (FIG. 6A). Since histidine isutilized as a source of both carbon and nitrogen in bacteria, the hutoperon is likely to be regulated by both vegetative (o⁷⁰ family) ando^(−N)-specific promoters (27), and therefore the occurrence of ₀,Npromoter might be physiologically important.

[0067] Expression of hutU gene in E. coli. Since the hutU gene of P.syringae contains some common features, including a long 5′-UTR ofcold-inducible genes observed in E. colt, it was interesting to examinethe temperature-dependent expression of the gene in the mesophilicbacterium. The expression of the ImtU gene in E. coli was examined bytransforming the bacterium with a multicopy plasmid (phlSO) thatcontained a 2.6-kbp DNA fragment with the structural gene for urocanaseand its upstream regulatory sequences from P. syringae. The cellextracts prepared from the £. coli grown at 37° C. exhibited a modestactivity of the enzyme (8.3 u,mol min′″ mg of protein″¹). However, theextracts prepared from the cells grown at a lower temperature (15° C.)did not contain any detectable urocanase activity. Thus, it appears thatthe hutU gene of P. syringae is not expressed in E. coli at the lowergrowth temperature. Whether the lack of expression is due to the absenceof cognate regulatory sequences and factors for transcription or to theinability of translation of the hutU mRNA at the lower temperature in E.coli remains unknown.

[0068] Discussion

[0069] The present study was undertaken to determine and analyze the DNAsequences of the hutU gene and its promoter region in order to identify,if possible, the putative regulatory c<< elements for the regulation ofthe hut operon in the psychro-trophic bacterium P. syringae.Additionally, it was thought that an analysis of the deduced amino acidsequence of the enzyme urocanase (the hutU-encoded product) from P.syringae, in comparison with the enzyme from mesophilic P. putida, mightprovide the clue to the nature of substitution of amino acids in a coldactive enzyme. An earlier study had demonstrated that a urocanase fromthe psychrophilic P. putida A.3.12 could retain, at 0° C., 30% of itsmaximum activity seen at 30° C. (21). The urocanase in P. putida is ahomodimer that contains tightly bound NAD* in each subunit, where anintact NAD* is essential for its catalytic reaction. Mechanistically,the enzyme is unique for the fact that the NAD* in this case does notfunction as a simple redox reagent but as an electrophile for thecatalytic addition of water to the urocanate for its conversion intoimidazolone propionic acid (7).

[0070] Analysis of the genetic organization and promoter region of thehutU in P. syringae. The organization of the genes in the hut operon isknown to be variable among gram-positive and gram-negative bacteria. Thepresent study indicates that the organization of the hut operon is alsovariable within the Pseudomonas species (FIG. 1B). The analysis alsoindicates that the nature of the permease gene within the hut operon isvariable. For example, Antarctic P. syringae has the hutP gene encodingpermease belonging to transporter class (TC) 2.A.39.1 (family NCS1),which is different from the hutT encoding a transporter of a differentclass (TC 2A.1.6.4, family MFS) observed in P. Putida (38). Thepathogenic P. aeruginosa has both the hutP and the hutT within the hutgene cluster. The published genetic study in P. putida had earlierfailed to locate any permease or transporter gene in the operon (19,20). A recent study in Sinorhizobium meliloti has demonstrated that thehistidine degrading hut H gene is linked to a histidine transporter ofATP-binding cassette type (9). Thus, there seems to be a randomrecruitment of the histidine transporters during the microbial evolutionof the histidine degradation pathway.

[0071] The regulation of the hut operon, which had mainly been studiedin three gram-negative bacteria, including Salmonella sp., K. aerogenes,and P. putida, and one gram-positive bacterium (B. subtilis) had alsobeen found to be variable (16, 26). For example, in B. subtilishistidine was found to be the main inducer of the operon, while in P.putida urocanase was found to be the inducer. Similarly, a positiveactivator HutP was observed to be the main regulator of the operon in B.subtilis, while the repressor HutC was found to be the main negativeregulator of the operon in P. putida. The regulator of the operon ishowever, complex in both systems for it is subjected to both carboncatabolite repression and nitrogen metabolite regulation in both of theorganisms. The temperature dependent expression of the operon in thecold-adapted bacterium P. syringae might further add to the complexityof the regulation of the operon.

[0072] In order to investigate the mechanism of temperature dependentregulation of the operon, the promoter region of hutU i.e., the firstgene of the operon in P. syringae, was been identified here by primerextension analysis. It appears that the mRNA for the hutU gene has along 5′-UTR that is a characteristic feature of many cold-induciblegenes of mesophilic bacteria, including E. coli. It also appears thatthe promoter region of the hutU of P. syringae contains various putativecis-acting regulatory elements that have been characterized earlier inmesophilic bacteria. However, the locations of these elements are atvariance with the known positions. For example, the repressor HutCbinding site, which is known to overlap either with the −35 sequence(e.g. in P. putide) or with the region between the −10 and −35 sequences(e.g., in K. aerogenes) of the hut promoters, has been found at the +143base (with respect to the 4° C. specific +1 site) and at the +95 base(with respect to the 4 or 22° C. specific common +1 site) region of the5′-UTR of the hutU gene of P. syringae (FIG. 6A). Similarly, a putativebinding sequence [ATA-(N₆)-TAT] for the positve activator NAC (fornitrogen assimilation control) rpotein (41) is located downstream of thetranscription sites (data not ehsonw). Interestingly, the HutC and NACbinding sites mentioned above overlap each other in P. syringae, thesignificance of which is not yet clear. As pointed out above, the CAPbinding sequence is also located downstream of the transcriptioninitiation sites of the operon in the cold-adapted bacterium (FIG. 6A).

[0073] The identification of a unique sequence CAAAA at the −10 site ofthe hutU promoter of P. syringae is interesting. This sequence isprobably important for the initiation of transcription at both low andhigh temperatures (e.g. 4 and 22° C.). An extra CAAAA sequence that isobserved a half turn away (5 hp) from the DNA helix in the region of the4° C. specific transcription start site might also be important forincreased expression of the gene at lower temperatures. The occurrenceof a putative o^(−N) (o⁻⁵⁴) specific promoter sequence, GG-(N₁₀)-GC, anda corresponding primer-extended product of RNA suggests that the operonmight be transcribed by RNA polymerase containing both vegetative sigmaand nitrogen regulatory sigma factors, depending upon the environmentalsignals. The observation of five to six primer-extended products of RNAfrom the hutU gene might also be a reflection of the complex regulatoryprocess of the operon.

[0074] It is also interesting that the putative cold-box sequenceobserved in the 5′-UTR of the cold-inducible genes of mesophilic E. coliis present in the coding region of hutU mRNA of P. syringae (FIG. 6).Whether this sequence has any role in P. syringae at lower temperaturesis a matter of conjecture. Recently, it has been shown in E. coli thatthe Y-box sequence (5′-ATTGG-3′) or its inverted repeat 5′-CCAAT-3′)might be important for the regulation of cold-inducible genes by anantitermination mechanism (5). In the psychrotrophic Y. enterocolitica,the induction of the pnp gene encoding polynucleotide phosphorylase atlow temperatures has also been shown to be regulated by the Y-boxsequence located 230 bp upstream of the translation start site (18). Thepromoter region of the low-temperature inducible gene icdII forisocitrate dehydrogenase in the psychrophilic bacterium Vibrio sp.strain AEB1 also contains a CCAAT sequence 2 bp upstream of the −35 siteof the promoter that is essential for low-temperature induction (37).The −10 and −35 sequences of the promoter (TAACTA and TTATAG,respectively) in the bacterium were however, not novel in any sensecompared to other housekeeping genes. The analysis of the present study,however, failed to show any Y-box sequence in the proper context of thepromoter, a 5′-ATTGG-3′ sequence observed 185 bp downstream of the GTGinitiation codon of the HutU (FIG. 6A).

[0075] In order to identify any other putative regulatory elements inthe promoter region of hutU, the potential secondary structural elementswere also examined. One such structure with a dyad symmetry (AG=−16.6keal) is located 70 nucleotides upstream of the transcription startsite. Such a hairpin loop structure can potentially function as atranscription stop signal for the upstream hutC or as a regulatoryelement for transcription of the hutU gene. Analysis of the urocanasesequence of P. syringae. The deduced amino acid sequence of theurocanase from P. syringae, compared with the sequences of the enzymehomologues from other mesophilic bacteria including K. aerogenes. A.rhizogenes, B. subtilis, S. coelicolor and D. radiodurans, did not showany obvious preference for any specific substitution of amino acids inthe protein due to the low-temperature adaptation. The active site ofthe enzyme in these bacteria (FIG. 2) has an almost identical sequenceFQGLPARICW, including the essential cysteine of the mesophilic P. putida(25). The two conserved cysteines (C-410 and C-354 of P. putida) arepresent in all of them. All of these enzymes have also a distinctsignature motif (GXGX₂GX₁₀G) of Rossmann fold, including an equivalentacidic residue at the end of the β-strand of the α/β-fold (FIG. 2).However, multiple alignment of the amino acid sequence suggests that theurocanase has two distinct diverged branches that might be related tothe phylogenetic origin of gram-negative and gram-positive bacteria(FIG. 3). The cold sensitivity of many cold-labile enzymes, includingNAD⁺specific glutamate dehydrogenase, from various bacteria have beendescribed to the ready dissolution of the monomeric subunits at a lowertemperature as a result of weakening hydrophobic bonds (22). Since thehydrophobic bonds stabilize the quaternary structures of proteins andsince the enzymes from cold-adapted bacteria have in general reducedhydrophobic interactions to acquire flexibility for functioning at lowertemperatures, it might be suicidal for cold active dimeric andoligomeric proteins to adopt a similar strategy. This could be one ofthe reasons why there are not many substitutions at the level of primarysequence in the dimeric enzyme urocanase from mesophilic P. putida andpsychrotrophic P. syringae. In fact, a recent study shows that thehydrophobic character of the homodimeric enzyme malate dehydrogenase ofthe psychrophilic bacterium Aquaspirillium arcticum is similar to thatof the enzyme from the thermophilic bacterium Thermus flavus (24).However, three major differences were noticed in the psychrophilicenzyme that were implicated in the efficient catalysts at lowertemperature. The differences include (i) an increased relativeflexibility at the active-site region of the enzyme; (ii) favorablecharge distribution, such as more positive potential around thenegatively charged substrate (oxaloacetate) binding site and decreasednegative potential around the cofactor NADH-binding site; and (iii)reduced intersubunit ion pairs and decreased buried surface area in thedimeric interface of the enzyme (24). A Similar structure-function studyon the urocanase of P. syringae would be useful to substantive thegenerality of these findings.

[0076] In conclusion, the present study shows that the −10 and −35characteristics of the promoter in P. syringae are unique. Theoccurrence of multiple in CAAAA might be important for low-temperaturemelting of the promoter. The present study also reflects the possiblecomplexity and uniqueness of regulation in the operon of P. syringae dueto the presence of many putative regulatory cis elements that arelocated downstream rather than in the usual location upstream of thetranscription start site. The present study also identifies two distinctclusters of urocanase sequences among the bacteria that might be relatedto their origin or lineage. The identification of a Rossmann told forNAD⁺ binding is also important for future modeling of the enzyme sincethis site is presumed to be different from other NAD-requiring enzymesfor its inaccessibility without denaturation of the urocanase.

1. A DNA sequence from nucleotide 2961 to 3600 of the upstream region ofcold-inducible hutU region of the Antarctic Psychrotrophic BacteriumPseudomonas Syringae of accession No.AF326719, comprising promoterelements and other regulatory sequences, with unique ‘CAAAA’ nucleotidesequence at −10 site of multiple transcription start sites and usingsaid promoter to express genes of interest in the said bacterium attemperature as low as 40° C. and using the said bacterium withgeneration time ranging between two and half to three hours, as a systemto produce low temperature labile proteins of pharmaceuticalsignificance.
 2. A sequence as claimed in claim 1 wherein, said promoterhas two transcription initiation sites with low temperature (4° C.), andcommon low and high temperature (4° C. and 22° C.) specificity.
 3. Asequence as claimed in claim 1 wherein, the amount of transcriptsproduced at 4° C. from the hutU gene and hut operon is about 20-foldhigher than the amount present at 22° C. during the steady-state growthof the said bacterium.
 4. A sequence promoter as claimed in claim 1wherein, the said gene is inducible upon a downshift of temperature from22 to 4° C.
 5. A sequence as claimed in claim 1 wherein, HutU is theOpen Reading Frame (ORF) of hutU gene.
 6. A sequence as claimed in claim1 wherein, amount of mRNAs from the hutU operon increased only about twoto three folds on temperature downshift of the said bacterium from 22 to4° C.
 7. A sequence as claimed in claim 1 wherein, amount of transcriptsproduced at 4 and 22° C. or from “cold-shocked” cells after a shift ofthe culture from 22 to 4° at different time points of 0, 0.5, 1, 2, and3 hours after that shift.
 8. A sequence as claimed in claim 7 wherein,the amount of mRNAs is at maximum by 2 hours after the shift anddecreased subsequently.
 9. A sequence as claimed in claim 1 wherein, 4°C. specific transcription start site starts with a G, which is 219nucleotides upstream of the translation initiation codon GTG of the HutUORF.
 10. A sequence as claimed in claim 1 wherein, the commontranscription start site for both low and high temperature is located 39nucleotides from the low temperature transcription start site.
 11. Asequence as claimed in claim 1 wherein, the −35 sequence in the promoterregion of the 4° C. specific transcript is TGTTAC.
 12. A sequence asclaimed in claim 1 wherein, the −35 sequence in the promoter region ofthe 4 and 22° C. common transcript is CCTGCG.
 13. A sequence as claimedin claim 1 wherein, the 4° C. specific transcript has second CAAAAsequence at the −15 position.
 14. A sequence as claimed in claim 13wherein, the second CAAAA nucleotide sequence at low temperaturetranscript is important for increased expression of the gene at lowertemperature.
 15. A sequence as claimed in claim 1 wherein, sequence atthe upstream of the GTG translational start codon of the hutU genecontain HutC repressor binding motif CTTGTATGTACAAG.
 16. A sequence asclaimed in claim 1 wherein, catabolite activator protein (CAP) bindingsequence, AAGTGTGCGTCGACCCTCTTGT, is located 35 nucleotides upstream ofthe GTG translation initiation codon of the hutU-ORF.
 17. A sequence asclaimed in claim 16 wherein, said Catabolite Repressor acts astranscriptional roadblock for RNA polymerase.
 18. A sequence as claimedin claim 1 wherein, a conserved “cold-box”-like sequence, TTGATGAACAACC,is located 123 nucleotides downstream of the translation initiationcodon of HutU-ORF.
 19. A sequence as claimed in claim 1 wherein,nitrogen regulatory σ^(N) promoter element, GGCCGCTTACTTGC, is located81 nucleotides upstream of the translation start site of the HutU-ORF.20. A sequence as claimed in claim 1 wherein, said HutU gene can not beexpressed in E. coli at temperature of about 15° C. and below.
 21. Asequence as claimed in claim 1 wherein, the Shine-Dalgarno (SD)sequence, GAGGA, is located 12 nucleotides upstream of the translationinitiation codon GTG of the hutU ORF.
 22. A sequence as claimed in claim1 wherein, the cold-shock protein binding sequence, ATTGG, is located186 nucleotides downstream of the translational initiation codon GTG ofhutU ORF.
 23. A sequence as claimed in claim 1 wherein, regulatorysequence GCGAGCTCTTGAATGCGGCCACCAAGAGCTCGC, having hairpin loopstructure is located 70 nucleotides upstream of low temperaturetranscription start site.
 24. A sequence as claimed in claim 23 wherein,change of free energy (ΔG) for the said loop is about −16.6 kcal.
 25. Asequence as claimed in claim 23 wherein, said loop functions as atranscription stop signal for the upstream hutC gene.
 26. A sequence asclaimed in claim 23 wherein, said loop functions as a regulatory elementfor transcription of the hutU gene.
 27. A method of cloning andexpressing cold-inducible hutU gene from the Antarctic PsychrotropicBacterium Pseudomonas Syringae, said method comprising steps of: d.cloning 2.4 kbp Pst DNA fragment containing hutU gene, and threeoverlapping fragments containing upstream 3.5 kbp Eco-RI-Kpn I fragment,downstream 2.54 Kbp SalI and 1.2 Kbp Pst I fragments into pUC19, e.sequencing the clone, and f. expressing the sequence clone.
 28. A methodof expressing gene encoding low-temperature labile proteins of interestusing DNA sequence of claim 1, said method comprising introducing saidsequence at the upstream region of the gene, and expression the saidgene, obtaining protein of interest.